Cyanobacteria are an ancient and diverse class of oxygenic photosynthetic bacteria characterized by their ability to use light energy to split water into oxygen and reductant, which is subsequently consumed in dark reaction of photosynthesis. Some cyanobacteria are also able to reduce atmospheric dinitrogen gas to ammonium (N2 fixation). Nitrogen fixation and oxygenic photosynthesis, however, are intrinsically incompatible, because nitrogenase, the enzyme responsible for reduction of N2, is inactivated by minute concentrations of oxygen. Certain diazotrophic (nitrogen-fixing) cyanobacteria, e.g., the genera Anabaena and Nostoc in the family Nostocaceae, overcome this incompatibility by differentiating specialized, microxic (low oxygen concentration) cells at intervals within chains of other cells. Nitrogen fixation is confined to these specialized terminally differentiated cells, called heterocysts, and oxygenic photosynthesis takes place in the vegetative cells. Some other filamentous diazotrophic cyanobacteria, such as Trichodesmium spp. have evolved similar microoxic cells for nitrogen fixation without visible differentiation into heterocysts (Berman-Frank et al. (2001) Science. 294: 1534-1537). An alternative to the spatial separation strategy is the temporal separation of photosynthesis and nitrogen fixation found in unicellular cyanobacteria such as Cyanothece sp. ATCC51142, Gloethece and Synechocystis sp. WH8501 (Berman-Frank et al. (2003). Research in Microbiology. 154:157-164). The diurnal separation of the two incompatible biochemical pathways allows the cyanobacteria to photosynthesize during the day and fix nitrogen at night. Unicellular cyanobacteria achieve nitrogen fixation at night by substaining a high rate of respiration at night, thereby creating a microoxic environment inside the cell for nitrogenase to function (Schneegurt et al. (1994). Journal of Bacteriology. 176: 1586-1597).
The heterocyst achieves a near anoxic state by at least three means. Although heterocysts have a photosystem I that they use to generate ATP, they lack O2-producing photosystem II, which is dismantled during heterocyst differentiation, so that the heterocyst need contend only against O2 produced by neighboring vegetative cells and that dissolved in the environment. Heterocysts also comprise a specialized envelope that limits the influx of gases. Two layers within the envelope have been implicated in O2 protection: an inner layer composed of a hydroxylated glycolipid and an outer layer of polysaccharide. Further, much of the O2 that overcomes these barriers is consumed by the high oxidase activity associated with heterocysts.
The heterocyst forms from vegetative cells. Within one or two generations, vegetative cells deprived of a source of nitrogen differentiate into N2-fixing heterocysts. The program of development begins with sensing nitrogen deprivation and culminates in the expression of the N2 fixation apparatus in the mature heterocyst, utilizing an ordered sequence of events.
Due to the absence of photosystem II, the heterocyst is dependent on adjacent vegetative cells for reduced carbon, just as the vegetative cells are dependent on heterocysts for reduced nitrogen. Nitrogen fixed within the heterocyst as ammonium is first converted to glutamine and then passes as amino acids to adjacent vegetative cells. In return, energy in form of fixed carbon flows from vegetative cells to heterocysts.
The expression of oxygen sensitive proteins in heterologous cells, particularly where the cells can be grown in ambient oxic conditions, is of great interest. The present invention provides such methods and compositions.